P464: COMPARED TO CLASSICAL CYTOGENETICS OPTICAL GENOME MAPPING (OGM) DETECTS MULTIPLE ADDITIONAL STRUCTURAL CHROMOSOMAL ABERRATIONS IN PEDIATRIC ACUTE MYELOID LEUKEMIA (AML)
Authors: J. Suttorp, J. L. Lühmann, D. Steinemann, D. Reinhardt, N. von Neuhoff, M. Schneider
Journal: HemaSphere (2022)

Background: AML is the 2nd most frequently diagnosed blood cancer in children affecting approximately 130 patients annually in Germany. Pediatric AML is a heterogeneous malignancy that has multiple genetic aberrations. This is relevant for classification, prognosis and selection of optimal therapy. To detect these alterations established procedures (karyotyping, FISH, RNA-based techniques) are used with well-known limitations (resolution of karyotyping and FISH, respectively; cell cultivation is necessary to generate metaphases; cost-intensiveness; necessity of experienced and intensively trained staff). OGM is an emerging technique addressing these limitations. Based on extraction of ultra-high weight DNA followed by fluorescent labelling at the sequence CTTAAG a barcode-like pattern of the genomic DNA is created. The DNA strands labelled in this way are stretched and drawn as single ultra-long fragments through nanochannels. The results are translated into molecule maps which are compared to the reference genome (resolution down to 500 bp) allowing the identification of genetic alterations. This method combines high resolution with picturing almost whole chromosomes without cell cultivation. Aims: The aim of this study was to test to what extend OGM might supplement classical cytogenetics (CCG) to detect risk defining genetic aberrations at initial diagnosis in pediatric patients with AML. Methods: We analyzed 24 specimen of pediatric AML, MPAL and bilineage leukemia patients by OGM starting from stored frozen material (bone marrow, peripheral blood) obtained at initial diagnosis. Results of OGM were compared with karyotyping and FISH. Primer walking and breakpoint spanning PCR were used to validate newly detected aberrations by OGM in selected cases. Applying breakpoint-spanning PCR, validated aberrations were used as markers for assessment of minimal residual disease (MRD) in selected cases during AML treatment. Results: Overall, we detected discrepant results between CCG and OGM in 17/24 (70%) cases including 9 cases in which karyotyping detected aberrations which were not found by OGM. However, these aberrations were not relevant for risk stratification. In general, focusing on genetic aberrations important for risk stratification the results between CCG and OGM were concordant in 23/24 (96%) cases. In a single case OGM was able to detect a high-risk aberration which was missed by CCG. In total, 33 previously unknown genetic alterations were identified by OGM. Out of these four newly detected variants (2 deletions, 2 translocations) were validated by PCR. The translocations t(2;12) and t(8,12) both affected gene ETV6 on chr. 12 with different fusion partners (chr. 2: AC064875.1; chr. 8: NSMCE2). In both translocations sequences at the breakpoints were determined. Two deletions affecting genes RNF157 on chr. 17 and MEF2B on chr. 19 were validated. These aberrations had not been described before in pediatric AML. In 2 cases - both without a previously identified MRD marker – the newly detected translocations by OGM were used for MRD monitoring. Summary/Conclusion: OGM considerably expands the range of techniques to optimize the diagnosis of pediatric AML and has much potential to omit limitations of classical cytogenetics. Furthermore, the results will contribute to a better understanding of the leukemogenesis of pediatric AML. In addition, OGM offers the possibility to identify new aberrations which can serve as patient specific MRD markers especially in cases without a previously known MRD marker.